In bacteria of the genus Rhizobium, nitrogenase synthesis is normally repressed under free-living conditions and is induced only within a complex symbiosis formed mostly with leguminous plants. R. trifolii is an example of a fast-growing Rhizobium with a narrow host range and cannot normally be induced to fix nitrogen in culture. In contrast, a Parasponia Rhizobium species has been isolated and this species is a slow-growing organism with a very broad host range capable of an effective symbiotic relationship with a broad variety of tropical legumes as well as the non-legume Parasponia (Ulmaceae) (Trinick, M. J. (1980) J. Appl. Bacteriol. 49: 39-53). Parasponia Rhizobium can be induced to fix nitrogen in culture although the level of this fixation is about 100-fold less than can be obtained from the free-living bacterium Klebsiella pneumoniae. Other slow-growing Rhizobia include the commercially significant R. japonicum, which nodulates soybeans.
The genetics of biological nitrogen fixation have been well characterized in the free-living organism Klebsiella pneumoniae. The structural genes for nitrogenase (nifH, nifD and nifK encoding the Fe-protein subunit and the .alpha. and .beta. subunits of the Mo-Fe protein, respectively) have been mapped both genetically and physically (Kennedy, C. et al. (1981) In Current Perspectives in Nitrogen Fixation (eds. Gibson, A. H. and W. E. Newton) Australian Acad. Science, Canberra, pp. 146-156; and Reidel, G. E., Ausubel, F. M. and F. M. Cannon (1979) Proc. Nat. Acad. Sci. U.S.A. 76: 2866-2870). Cloned DNA fragments carrying these sequences have been shown, by Southern blot analysis, to hybridize to homolgous sequences in a wide range of nitrogen fixing organisms, including Rhizobium (Ruvkun, G. B. and F. M. Ausubel (1980) Proc. Nat. Acad. Sci. U.S.A. 77: 191-195).
In spite of the ecological diversity of nitrogen fixing organisms, the physiological structure of the nitrogenase enzyme complex appears to be very conserved. In all cases where the enzyme complex has been purified, two proteins are present. The larger protein (dinitrogenase) contains molybdenum, iron and acid-labile sulfur, and carries the binding site for nitrogen and contains two subunit proteins .alpha.- and .beta.-coded by the nifD and nifK genes respectively. The smaller protein (dinitrogenase reductase) contains iron and acid-labile sulfur, and is required for the reduction of the dinitrogenase and for the binding of MgATP used in this reduction. The dinitrogenase reductase is coded by the nifH gene. Chemical and spectral analyses of the purified protein components support a conservation of protein structure between organisms (Scott, K. F., Rolfe, B. G. and J. Shine (1981) J. Mol. Appl. Genet. 1: 71-81). In some cases the structures are sufficiently similar to allow formation of active hybrid enzymes between purified components, e.g., Azotobacter vinelandii and Klebsiella pneumoniae (Eady, R. R. and B. E. Smith (1979) In: A treatise on dinitrogen fixation I, II, eds. Hardy, R. W., Bottomley, F. and R. C. Burns, New York, Wiley Press pp. 399-490). Not surprisingly, therefore, the region of the nif operon coding for dinitrogenase reductase and dinitrogenase .alpha.-subunit (nifH and nifD) shows homology at the nucleic acid sequence level with the corresponding sequences in at least 19 other bacterial strains (Ruvkun, G. B. and F. M. Ausubel (1980) Proc. Nat. Acad. Sci. U.S.A. 77: 191-195). Although this conservation of structure is generally true, significant differences between nitrogenases from different organisms also exist as can be shown by variable stability following purification and by the fact that active hybrid complexes do not form in all cases (Eady, R. R. and B. E. Smith (1979) supra).
A DNA fragment carrying the Klebsiella pneumoniae nifK, nifD and nifH genes has been isolated from the nif strain UNF841(Tn5::nifK) (Cannon, F. C. et al. (1979) Mol. Gen. Genet. 174: 59-66) and cloned into the Escherichia coli plasmid pBR325. The nucleotide sequences of the nifH gene and of 622 nucleotides of the nifD gene were determined (Sundaresan, V. and F. M. Ausubel (1981) J. Biol. Chem. 256: 2808-2812; Scott, K. F., Rolfe, B. G. and J. Shine (1981) supra). In addition, the DNA sequence of the nifH gene from Anabaena 7120 has been determined (Mevarech, M., Rice, D. and R. Haselkorn (1980) Proc. Nat. Acad. Sci. U.S.A. 77: 6476-6480). A comparison of the two sequences demonstrates two interesting features: (1) There is very little homology between the two sequences at the nucleotide sequence level although a few stretches (up to 25 bp) are conserved, accounting for the observed interspecies homology of the nif genes (Ruvkun, G. B. and F. M. Ausubel (1980) supra); (2) In general, the promoter regions show very little sequence homology with the exception of a short region likely to be involved in common functions, e.g., RNA polymerase recognition.
In contrast, a comparison of the amino acid sequences of the dinitrogenase reductase and of the first 207 amino acids of the .alpha.-subunit of dinitrogenase of the two species and of another species show a much greater conservatism. The three species used in this comparison are Klebsiella pneumoniae (Kp); Anabaena 7120 (Ab); and Clostridium pasteurianum (Cp) (Tanaka, M., Haniu, M., Yasunobu, T. and L. Mortenson (1977) J. Biol. Chem. 252: 7093-7100). The Kp and Cp proteins share 67% amino acid sequence homology, Kp and Ab proteins share 71% homology, and the Cp and Ab proteins share 63%. This amino acid sequence homology is not spead evenly throughout the protein. Some regions are virtually identical--90% to 95% homology), while other regions are only weakly conserved (30-35% homology). The structural conservation appears to be centered around the five cysteine residues common to all three Fe proteins. These cysteine residues are believed to be ligands to the active center.
Comparison of the N-terminal amino acid sequence of the .alpha.-subunit of dinitrogenase from Cp and Kp shows very little sequence homology in this region. This is in contrast to the very high conservation of amino acid sequence seen in the amino terminal region of the Fe protein. What little homology exists between Cp and Kp .alpha.-subunits is confined to regions around cysteine residues, as in the Fe proteins. These homologous regions are thought to be involved in the catalytic functions of the nitrogenase enzyme complex. Therefore, this structural conservatism is thought not to be the result of recent evolution and dispersal of the nif genes (Postgate, J. R. (1974) Sym. Soc. Gen. Microbiol. 24: 263-292) but, rather, is postulated to be related to a conservation of function.
The isolation of Klebsiella pneumoniae DNA which codes for the structural genes of the nitrogenase complex (Ruvkun, G. B. and Ausubel, F. M. (1980) Proc. Nat. Acad. Sci. USA 77: 191-195) has facilitated the identification of the corresponding structural genes of Rhizobium meliloti. The R. meliloti genes were found on an EcoRI fragment which was cloned in E. coli plasmid vectors (Ruvkun, G. B. and F. M. Ausubel (1981) Nature 289: 85-88). Further studies using fragment specific mutagenesis in E. coli and transfer of the mutations to the R. meliloti genome confirmed that the cloned fragment carries nif specific genes (Ruvkun, G. B. et al. (1980) Cold Spring Harb. Symp. Quant. Biol. 45: 492-497; Ruvkun, G. B. et al. (1982) Cell 29: 551-559). This cloned R. meliloti fragment has been analysed by the minicell technique and it was demonstrated that the nifH gene of R. meliloti was expressed in minicells of E. coli (Weber, G. and A. Puhler (1982) Plant Mol. Biol. 1: 305-320). In free living R. meliloti, the nifH gene is not expressed. Further experiments have identified a number of R. meliloti genes involved in symbiotic nitrogen fixation and a preliminary map of R. meliloti nif and fix genes was published (Puhler, A. et al. (1983) In: Advances in Nitrogen Fixation Research (ed. by Veeger, Newton). The Hague, Boston, Lancaster). This map contains the coding regions of the following R. meliloti genes nifK, nifH, nifD, fixA, fixB and fixC (FIG. 1). This figure gives a preliminary restriction map of the Rhizobium meliloti nif and fix genes and their promoters. In addition, the coding regions of the various nif and fix genes are shown by black arrows. The direction of transcription and translation is also shown by the arrows. nifK and nifD are presented as hybrid genes. The indicated promoters (p) were identified in E. coli. The nifH as well as the fixA promoter can be activated by the K. pneumoniae nifAgp protein. The abbreviations used in the restriction map are: C, ClaI; E, EcoRI; H, HindIII; P, PstI; S, SmaI; and X, XhoI.
Of special interest is the transcriptional regulation of the R. meliloti fix/nif region. In FIG. 1, two promoters are indicated; one is located next to nifH and the other is next to fixA. For both the nifH promoter and the fixA promoter it has been demonstrated that they can be activated in E. coli by the Klebsiella nifA gene product (Puhler, A. et al. (1983) see supra). The nifH and the fixA promoter are reading in opposite directions. Sundaresan, V. et al. [(1983) Nature 301: 728-732] and Sundaresan, V. et al. [(1983) Proc. Nat. Acad. Sci. USA 80: 4030-4034] identified and sequenced the nifH promoter and found some homology to the K. pneumoniae nifH promoter. Corbin, D. et al. [(1983) Proc. Nat. Acad. Sci. USA 80: 3005-3009] also identified the fixA promoter as well as the nifH promoter of R. meliloti. In contrast, the K. pneumoniae nifH promoter cannot be activated by the E. coli glnG gene product, whereas the R. meliloti nifH promoter can be activated by the E. coli glnG gene product. These observations imply that the activator and/or the promoter of nifH in Klebsiella pneumoniae and Rhizobium meliloti are different. Indeed, no endogenous activator of the nifH and fixA genes of R. meliloti was known.